Thrombospondin (TSP)-1 is an ~420KDa trimeric, multidomain glycoprotein that engages multiple endothelial cell (EC) receptors and elicits multiple EC responses. The zonula adherens (ZA) is a specialized structure that, through the catenins, couples the actin cytoskeleton to the cytoplasmic domain of vascular endothelial (VE)-cadherin, a surface receptor that mediates homophilic EC-EC adhesion. In human pulmonary microvascular endothelia, we have demonstrated that TSP-1 increases tyrosine phosphorylation of the ZA proteins, VE-cadherin, y-catenin and p120ctn, and opens the paracellular pathway. Prior protein tyrosine kinase (PTK) inhibition protects against TSP-1-induced barrier disruption whereas protein tyrosine phosphatase (PTP) inhibition enhances it. We now present evidence that TSP-1 activates the receptor PTKs, ErbB1 and ErbB2, and opens the endothelial paracellular pathway through this activation. Further, PTP(, a receptor PTP that regulates VE-cadherin tyrosine phosphorylation and the endothelial paracellular pathway, counter-regulates EGFR/ErbB2 activation. We propose the following Specific Aims: 1) To define the specific sequence(s) within TSP-1 required to open the tyrosine phosphorylation - responsive paracellular pathway in human pulmonary microvascular endothelia. 2) To elucidate the mechanism(s) through which TSP-1 activates EGFR/erbB2, including a) direct binding of its EGF-like repeats to the ectodomain of EGFR, b) stimulation of EC release of an EGFR ligand, and/or c) activation of another TSP-1 receptor(s) that transactivates EGFR/erbB2. 3) To determine the mechanism(s) through which TSP-1 activation of EGFR/erbB2 is coupled to increased tyrosine phosphorylation of ZA and/or other junctional proteins, ZA reorganization, and/or opening of the endothelial paracellular pathway. 4) To define the counter-regulatory role for PTP( in TSP-1-induced EGFR/ErbB2 activation. Understanding the mechanism(s) through which TSP-1 regulates EC-EC homophilic adhesion and the paracellular pathway has implications for angiogenesis within the context of wound healing, tissue remodeling, and tumor cell survival, as well as the movement of cells, macromolecules, and fluids from the bloodstream into extravascular tissues.